Experimental Study On Establishing The Tuberculosis Model In Chinese Rhesus Macaques

According to the World Health Organization report,approximately one third of the world's population is infected by Mycobacterium tuberculosis(M.tb).Due to limited protection of BCG and lack of high effective anti-tuberculosis drug,investigating new safe and effective vaccines and drugs that can prevent or cure all forms of tuberculosis has become a top topic for all the scientists who working on anti-tuberculosis.Animal models of tuberculosis infection could be a useful tool to understand the nature of the disease process,especially the specific information about the disease pathology and the immune response.In addition,standardized animal models could be an experimental platform which can be used to test new anti-TB agents or vaccines.In this study we established three nonhuman primate tuberculosis models and assessed their clinical and immunologic indexes as potential sensitive measures of disease burden.1.Establishment of a tuberculosis model in Chinese rhesus macaquesNonhuman primate model of tuberculosis is closed to humans' disease;it is very useful for studying pathogenesis or testing vaccines and drugs.Here a Chinese original rhesus macaque model of tuberculosis was established by three different challenge routes(bronchoscopic instillation,intratracheal instillation and nasal instillation)with two different doses(20 CFU and 100 CFU)of M.tuberculosis H37Rv strain.Macaques were grouped and followed clinically post infection,including physical examinations,Clinical assessment,Chest X-rays,ELISAs and ELISPOT assays.Microbiologic and pathologic events were assessed 24 weeks post infection.From these studies,we conclude that the macaques can be successfully infected via three challenge routes,and the low infective dose(20 CFU)can achieve a subclinical model of tuberculosis.The traditional clinical indexes were showed to correlate with protection,and imaging offered a potentially reliable correlate.But immunological measures were found not to correlate with protection.These results suggest Chinese rhesus macaques can be used as a model for tuberculosis.2.Establishment of a natural tuberculosis infection model of Chinese rhesus macaquesHuman TB is transmitted through M.tb aerosols from infected people.The majority(90%)of infected people develop latent TB infection.Because of the similarities in M.tb infection between macaques and humans,macaque models have been extensively used for TB research.However,the majority of artificially infected macaques develops acute/active TB disease,and dies within a short time period.Therefore,it is necessary to establish a latent M.tb infection macaque model through natural infection such as aerosol transmission.This model is not only useful for understanding the immunopathogenesis of M.tb latent infection,but also provides a clinically valuable macaque model for the development and evaluation of new vaccines and drugs against TB.In an initial experiment,six Chinese-origin rhesus macaques were located in a modified cages system in which the air can cycle within the cages of the study animals.Two animals were infected via intra-tracheal instillation with M.tb(Erdman strain,34 CFU).The remaining four healthy animals were sentinel for transmission.Serial tuberculin skin test(TST)reactions and ELISPOT results were measured to determine M.tb infection.Physical and medical examination(Chest X-rays,CT scan,ESR and CRP in plasma)were performed monthly.All the four animals showed TST and ELISPOT positive at week 22,24,27 and 42,respectively.In addition,Chest X ray and CT scan showed the primary lesions in upper lobes of lungs,which differs from the artificial infection where lesions are seen in right lower lobes of lungs.In the second experiment,8 monkeys were involved by using the similar methods in the initial experiment.One monkey were successfully natural infected with M.tb at week 16 and maintained latent infection.It is feasible to experimentally establish natural M.tb transmission of Chinese-origin rhesus monkeys.3.SIV infection facilitates M.tb infection of rhesus macaquesTuberculosis(TB)is a common opportunistic infection and the leading cause of death for human immunodeficiency virus(HIV)-infected patients.Thus,it is necessary to understand the pathogenetic interactions between M.tb and HIV infection.In this study,we examined M.tb and/or simian immunodeficiency virus(SIV)infection of Chinese rhesus macaques.While there was little evidence that M.tb enhanced SIV infection of macaques,SIV could facilitate M.tb infection as demonstrated by X-rays,pathological and microbiological findings.Chest X-rays showed that co-infected animals had disseminated lesions in both left and right lungs,while M.tb mono-infected animals displayed the lesions only in right lungs.Necropsy of co-infected animals revealed a disseminated M.tb infection not only in the lungs but also in the extrapulmonary organs including spleen,pancreas,liver,kidney and heart.The bacterial counts in the lungs,the bronchial lymph nodes,and the extrapulmonary organs of co-infected animals were significantly higher than those of M.tb mono-infected animals.The mechanistic studies demonstrated that two of three co-infected animals had lower levels of M.tb specific IFN-y and IL-22 in PBMCs than M.tb mono-infected animals.These findings suggest that Chinese rhesus macaque is a suitable and alternative non-human primate model for SIV/M.tb coinfection studies.The impairment of the specific anti-TB immunity is likely to be a contributor of SIV-mediated enhancement M.tb infection.